The Establishment And Application Of A Semi-nested PCR Method To Detect A Novel Goose Parvovirus From Duck

Goose parvovirus is one member of Parvovirinae,it can lead ducklings and Mus covy ducklings to morbidity with small intestinal mucosal surface necrosis falls off an d forming small intestine embolism as the main characteristics.Since 2015,there have been domestic continuous reports of Cherry Valley Ducks and Peking ducks breaking out disease with a short beak and long tongue as the characteristic symptoms(BADS).Through isolation and identification by Chen et al.,it is confirmed that this virus be longs to a novel Goose Parvovirus from duck(NGPV).The aim of this study was to build a semi-nested PCR method to detection the NGPV,and carryed out an epide miology investigation of NGPV for Shandong&Jiangsu with the method,meanwhile w e carryed out an experiment of sequencing the complete genome of 2 separated strain s and carryed out the homologous analysis and phylogenetic tree analysis.1 The preliminary detection of epidemic materials from Shandong&JiangsuAccording to the data of Gene Bank published already,we designed the detection primers of GPV using DNASTAR.Lasergene.V7.1.We carryed out the detection with extracting DNA from the heart,liver,spleen,lung,kidney,thymus and bursa of Fabricius,pancreas,brain,bile 10 viscera tissues total of 1200 samples of of the positive rate of every batch and every viscera.we analyzed the specificity of the primers and statistics.Results showing that with the method the highest and the lowest positive rate of every batch of was 86.7% and66.7% and the average positive rate was 75.8%;and the highest and the lowest positive rate of every viscera was 62.6%(Liver)and 9.9%(Lung)without brain and the average positive rate was 34.5%.2 The establishment and applying of a semi-nested PCR method detecting NGPVAccording to 12 strains of GPV and 2 strains of NGPV sequences from Gene Bank published already,we designed 2 pairs of primers to detect NGPV with Lasergene.V7.1software.The primers included inside pairs of primers according to the unique sites based onthe outside primers of GPV and NGPV,which can augment gene of the two types virus,about 799bp;the inside primers can augment gene of novel Goose Parvovirus from duck about 148 bp.To acquire the highest efficiency,through exploring we confirmed that the best optimal annealing temperature is 58℃,the fittest concentration of magnesium 2 + is 1.5 m M(1.5 μL)and the most appropriate primer concentration is 1μL(1p M).The method of Semi-nested PCR has a good specificity which shows negative to Du CV-1 and Du CV-2 and DVE and E.coli and SE and Ra that are common disease to ducks,and this method is also having a wonderful sensitivity,which can check out 100 copies nucleic acid of the virus.We carryed out an epidemiology investigation of the NGPV to the 1200 samples from Shandong and Jiangsu with the built Semi-nested PCR method,we still gave a full detection to the heart,liver,spleen,lung,kidney,thymus 10 internal organs total 1200 samples,then count the positive rate of every batch and each viscera and made a comparative analysis with the data from detection with normal PCR.Results showed that the detection with the semi-nested PCR had a higher infection rate of NGPV of every batch,and the highest rate reach to 93.3%,the lowest reaches was to 73.3% and the average rate reached to 80.8%;for each organ’s detection,the highest rate and the lowest rate was 93.8%(Liver)and23.7%(Lung)of every batch,the average detection rate was 55.8%,the detection rate was obviously improving compared with the ordinary PCR detection,but we still detected nothing from brains.The epidemiology investigation indicated that NGPV was seriously epidemic in Shandong and Jiangsu,which should be given high attention.2 Two NGPV strains complete genome sequencing and gene evolution analysis of SD-01 and SD-02According to 12 strains of GPV and 2 strains of NGPV sequences from Gene Bank published already,we designed 5 NGPV sequence pairs for complete genome amplification using DNASTAR.Lasergene.V7.1 software,synthesizing by Shanghai Sangon biological engineering co.LTD.According to 12 GPV strains and 2 strains NGPV sequences the Gene Bank published already,we designed 5 NGPV sequence primers for complete genome amplification using DNASTAR.Lasergene.V7.1 software,synthesizing by Shanghai Sangon biological engineering co.LTD.,we augmented the complete genome with 5 pairs of primer the sequenced,then put together,at last we obtained 2 unabridged genome.Then we gave ahomology analysis and evolutionary analysis of the 2 NGPVs with 12 GPV strains and 2NGPVs and 4 MDPVs The homology analysis results showed that SD-01 and SD-02 had a homology rate of 80.9% and 81.1% with a MDPV of FM strain,but had a rate of 92.2~97.0% and 99.1~99.6% with12 GPVs and 2 NGPVs.The evolutionary tree analysis results showed that the two separated strains belonged to GPV this big branch,and they were in a small branch with sdlc01 and QH15;We also gave a homology analysis to VP3 gene,finding that the homology analysis rate of SD-01 and SD-02 had the lowest homology rate of 92.2%with GDa GPV,and had the highest rate of 98.3% with 82-032 v,SD-01 had the same rate of99.8% with sdlc01 and QH15,but just 81.5% with MDPV-FM.We also conducted a homology analysis for amino acid sequences deduced by VP3,finding that both SD-01 and SD-02 had the lowest homology rate of 96.6% and 96.4% with GDa GPV strain,and SD-01 had the highest homology rate of 99.7% with B and VG321,and had a homology rate of100% and 99.6% with sdlc01 and QH15 each,and SD-02 have a rate of 99.8% and 99.4%with QH15.Evolutionary tree analysis shows that SD-01 and SD-02 are on different branchs,but on the same branch with sdlc01 and QH15,therefore had a far relationship with other GPVs.